We have previously obtained and elucidated the precise structure of a highly branched 1,3-β-D-glucan (with 6-monoglucopyranosyl side chains), Aureobasidium pullulans-fermented β-D-glucan (AP-FBG), from the fungus A. pullulans. However, the mechanism(s) of the effects of AP-FBG on in vitro mouse primary cells have not been analyzed in detail. Herein, we report that the induction of cytokines by AP-FBG was dependent on the existence of a granulocyte macrophage colony-stimulating factor (GM-CSF); this is similar way to be a typical 1,3-β-D-glucan from Sparassis crispa (SCG), which is a 1,3-β-D-glucopyranosyl backbone with single 1,6-β-D-glucopyranosyl side branching units every three residues. In other words, the production of cytokines in DBA/2-mouse-derived splenocytes by AP-FBG was completely hampered by an anti-GM-CSF neutralizing monoclonal antibody. Furthermore, the addition of exogenous GM-CSF to C57BL/6-derived splenocytes, which are less sensitive to AP-FBG, induced the production of cytokines by AP-FBG. Therefore, GM-CSF is indispensable for the induction of cytokines by AP-FBG in mouse-derived splenocytes. This finding has provided a new insight into our understanding of the actions of β-D-glucan but will also aid in the design and development of more effective β-D-glucan agents.